Lecture 1 Molecular Techniques Student.pdf

Biology 201 Dr. Torah Hunt Topic I – Studying Cells - Techniques A little note about your notes! For each lecture you will receive an outline of what will be covered on the course website. These are no...

Biology 201 Dr. Torah Hunt Topic I – Studying Cells - Techniques A little note about your notes! For each lecture you will receive an outline of what will be covered on the course website. These are not complete notes. Please bring the posted figures and outlines to class as we will be filling them out. Note: You must attend class to get a full set of notes, for any missed classes you can come see me and copy out the missed lectures. Introduction: In this lecture we will be introduced to the concept of cells and the techniques used to study them. You should be able to analyze which technique is best for solving a given problem and be able to discuss why you chose that particular technique. Objectives: ✓ Classify the different cell types ✓ Outline the basic principles of microscopy ✓ Explain the different types of microscopy ✓ Describe the advantages and disadvantages of using stains to study cells ✓ Describe recent advances in studying and visualizing live cells ✓ Explain the purpose of differential centrifugation and sedimentation ✓ Outline the steps in a Western blot ✓ Be able to determine which technique would be best for a given cellular question Outline 1. What are cells? - Schleiden and Schwann (1839) – Cell Theory - 2 Broad groups of cells o Prokaryotic – Bacteria and Archaea o Eukaryotic 1 Biology 201 Dr. Torah Hunt - Can either be: o Unicellular ▪ organisms consisting of one cell. ▪ can be Prokaryotic (bacteria) or Eukaryotic (protozoa) o Multicellular ▪ often consist of differentiated cells that perform specialized functions. ▪ - How small are cells? o Plant cells – o Animal cells – o Bacterium – o Ribosome – o Microfilament – - What types of cells are there? o Humans have 200 different types of cells divided into 5 categories: 2 Biology 201 Dr. Torah Hunt 2. Looking at Cells – Microscopy - Concepts in Microscopy - We study cells using microscopy: o There are several types of microscopy we can use to visualize elements of the cell o An important thing to consider: the vast majority of aspects of the cell we care about are not visible to the naked eye, and sometimes not even with microscopes. We need to find both direct and indirect ways to visualize cell processes 2.1 Light Microscopy - The minimum distance that two objects have to be in order to be distinguished as separate = resolution - The resolving power of; Human Eye = 90 µm (0.09 mm); human hair Light Microscope = 200 nm (0.2 µm) Electron Microscope = 0.2 nm - All microscopes produce a magnified image of a small object - Total magnification is a product of the magnification of the several lenses that operate in a conventional light microscope – objective and ocular lens - Resolution – ability to see two objects as distinct - Formula for resolution – 3 Biology 201 Dr. Torah Hunt 2.2 Interference Microscopy - Phase Contrast Microscopy - Generates an image in which the degree of darkness or brightness depends on the refractive index of that region a) Light microscopy b) DIC c) Phase contrast - Differential Interference Contrast (DIC) Microscopy - Contrast is generated by differences in the index of refraction of the object and its surrounding medium 4 Biology 201 Dr. Torah Hunt 2.2.1.1 Fluorescence Microscopy - Very powerful technique for visualizing subcellular structures and even individual proteins 2.3 How do we get fluorescent molecules in the cell? 2.3.1 Immunofluorescence - Antibodies are immunoproteins made to detect a specific antigen - Then – two possibilities - Advantage: very specific and good resolution - Disadvantage: Requires fixed specimens = dead 5 Biology 201 Dr. Torah Hunt 2.3.2 GFP – Green Fluorescent Protein - The gene for a naturally fluorescing protein has been isolated and cloned - The gene for GFP can be added to the ends (usually) of proteins of interest to tack on a fluorescent tag in live organisms 2.3.3 Confocal Microscopy - Traditional fluorescence microscopy can be blurred from signals coming from above and below the plane of focus and can be difficult to view signals in thick specimens - Confocal microscopy uses a pinhole laser beam to excite only a small plane of the specimen with fluorescent light - Advantage: - Disadvantage 6 Biology 201 Dr. Torah Hunt 2.4 Electron Microscopy - Much higher resolution because it is a beam of electrons, not wavelengths of light - Requires fixed and sectioned cells (dead cells) - Two types – Scanning EM o Transmission EM - Approx 40,000 x better than light microscopy for theoretical limit of resolution - TEM – image depends on differential scattering of incident electrons by molecules in the preparation - SEM – same concept but now it measures amount of electron scatter off the surface of a heavy-metal coated object. 7 Biology 201 Dr. Torah Hunt 3. Studying Subcellular Components - Many studies on cell structure and function require isolated organelles and cellular structure - A cell homogenate is first prepared by: sonication, chemical lysis, pressurized homogenators among others a) Sample Preparation Tissues / Cells are first homogenized or disrupted to separate contents. Sample forced through narrow orifice, ultrasonic vibration, osmotic shock, enzymatic treatment, manual grinding. b) Differential Centrifugation (Figure 12A-1, Page 327) c) Equilibrium or Density Gradient Centrifugation 8 Biology 201 Dr. Torah Hunt 4. Studying Proteins - Proteins do everything in the cell. - Studying proteins allows us to further understand the inner workings of a cell - Several techniques are used in protein-based investigations I. Electrophoresis 9 Biology 201 Dr. Torah Hunt II. Western Analysis Detection of specific proteins in a gel using antibodies Gels are stained with generalized dyes that label all proteins, if detection of a specific protein is needed we can use an antibody to determine if that protein is present Example: HIV test involves a Western blot to detect if the HIV antibodies are present in the patient o Membrane containing HIV proteins is blotted with patients blood serum o If HIV-antibodies are present they will detect HIV proteins on the blot 10